PCT/FI2006/050323 published 18.1.2007, describes a modification of glycosylation of stem cells. The examples show reaction of human cord blood mononuclear cells, comprising hematopoietic stem cells, with sialyltransferase and fucosyltransferases in MOPS buffer and 150 mM NaCl and by sialidases in acetate buffer.
The “Sackstein” application (WO 2007/143204) indicates, and their corresponding publication in Nature Medicine, filed after the priority date of the above application, claims specifically reaction conditions without any divalent cations and the application specifically shows toxicity of MnCl2. Based on the prior art it would not appear useful to use toxic Mn2+ or other divalent cations Mg2+ or Ca2+-ions in the reactions.
WO 2008/011094 also by Sackstein describes the use of cytokines to stimulate glycosylation enzymes on hematopoietic stem cells. The present invention is directed to the use of non-toxic divalent cations, small molecules and supporting non-glycoprotein albumin in context of in vitro modification of especially adherent cell. It is realized that activation of cells by cytokines may also produce undesired differentiation or cell activation leading to negative activities in context of in vivo or ex vivo uses of the cell.
PCT/FI2008/050015, filed 18.1.2008, by the present inventors indicates that the reactions even with the adherent cells can be performed in the presence of Mg2+, which would be preferred for the activity of modification enzymes, especially sialyltransferase and fucosyltransferase. The example includes reactions to mesenchymal stem cells in α-MEM, which contains also other factors revealed useful by present invention. The present invention revealed that use of both Mg2+ and Ca2+ ions preferably with supporting factors is especially useful for producing optimally viable cells. The preferred conditions further include conditions to prevent aggregation and surface binding of cells, especially preferred shear force conditions.
PCT/FI2008/050015, filed 18.1.2008, includes enzyme tagging technologies. The present invention provides specific forms and methods for the tagging, including glycan tagging with multiple tags and limited lysine-specific tagging by biotinylation.
It is realised that previously published cell modification conditions without divalent cations are not optimal for cell status. It was revealed that the condition induced morphological changes of cells, including non-natural granularity of the cells which are likely to reduce the viability of the cells, though the cells were indicated to be alive according to Sackstein et al.
The present invention is further directed to the use of strong protease conditions producing unicellular cell suspension. It is realized that this is needed to produce effective glycosylation and optimal unicellular product for subsequent in vivo use. It is realized that the cell population produced under the Sackstein conditions with a low amount of protease might not result in a unicellular preparation.
The process described by Sackstein and colleagues produces an N-glycan-linked sialyl Lewis x (sLex) epitope, but essentially no other selectin ligands structures (Sackstein et al. Nature Med 2008, 14(2):181-187). The present invention, including a reaction condition with divalent cations, effectively produced the sLex on O-glycan type structures different from the modification indicated by Sackstein.
The present invention further provides methods for specific tagging and washing cells with glycosyltransferase inhibitors to remove the enzymes from cell preparations after modification reactions.
Changes of sialylation by desialylation and resialylation with specific sialyltransferases has been reported for red cells in order to analyze binding specificities of influenza virus (Paulson, J. et al.).
Partial desialylation and alpha-6-resialylation of human peripheral blood and bone marrow CD34+ cells, both non-adherent blood cells, has been reported, the peripheral blood cells having been released by GM-CSF and most of the subjects being under cancer therapy (Schwarz-Albiez, Reihard et al., 2004, Glycoconj. J. 21:451-459). The large variations in results may be due to therapy and GM-CSF. The method used did not reveal quantitation of sialic acid types, due to a limited specificity of the sialyltransferase used. The modifications of sialic acid would likely further affect the acceptor specificity of the sialyltransferase used and thus the structures labelled. The present invention is especially directed to α3-sialylation of the specific carrier structures.
Removal of NeuGc from pig xenotransplant tissue and resialylation by NeuAc and sialyltransferase has been also suggested (WO 2002/088351). That work was not directed to stem cells, nor human stem cells directed methods, nor were the methods used specified, although this is essential for applications in these cells. The xenotransplantation idea is not relevant to present invention due to tissue and species specificity of glycosylation. A patent application (WO 2003/105908) describes possible sialidase and sialyltransferase reactions for certain NK or lymphocyte cell lines. The results revealed that the reactions varied between the cell lines and were not predictable under the conditions used in the work. Further, the reaction conditions of sialyltransferase without CMP-sialic acid were not described by the inventors.
Use of an inhibitor after glycomodification reaction has been indicated, but the chemical nature of the inhibitors was not indicated (WO 2004/072306). There is no indication of use of an inhibitor, especially soluble acceptor which mimics competitive inhibitor in context of cells and sialidase reactions or glycosyltransferase reactions. Furthermore, no useful concentration ranges for the substrates has been indicated and the present inventors were first to reveal the need of removal of bound glycosyltransferases from enzymes.
Xia et al. (2004) Blood 104 (10) 3091-9 describes changing fucosylation of cord blood cell population without changing sialylation levels and possible usefulness of the modification in targeting of the cells to the bone marrow.